Cell proliferation and tissue invasion inhibitor

ABSTRACT

A composition of matter having activity as an inhibitor of cell proliferation .Iadd.and tissue invasion.Iaddend.is obtained by aqueous extraction of tissue having a high content of collagen and/or proteoglycans.

The invention described herein was made in the course of work under agrant or award from the Department of Health, Education and Welfare.

The present invention relates generally to a composition of matterhaving activity as an inhibitor of cell proliferation .Iadd.and aninhibitor of tissue invasion. .Iaddend.More particularly, it relates tomethods of preparing such composition, and to methods of inhibitingproliferation of cells utilizing such composition.

In accordance with the present invention, a substance having activity asan inhibitor of cell proliferation or, as it is sometimes referred toherein, an inhibitor of cell growth, is prepared by extractive methodsfrom tissue having a high content of collagen and/or proteoglycans, andpreferably from connective tissue. The extract may be treated so as toconcentrate the inhibiting substance. The resultant concentrate hasutility in inhibiting proliferation of cells, and particularly ininhibiting proliferation of fibroblasts and endothelial cells.

More particularly, in the preferred embodiment of the present invention,connective tissue is extracted with an aqueous extraction medium. Apreferred extraction medium includes a solute which does notirreversibly denature proteins or proteoglycans. One such preferredextraction medium comprises a 1.0-3.0 M aqueous solution of guanidinehydrochloride.

Examples of connective tissue which may be used in the preferredembodiment of the present invention include cartilagenous andligamentary tissues, vascular tissues, corneal tissues, dental tissuesand dermal tissues. The connective tissue is placed in condition forextraction by mincing, comminuting, or otherwise treating it to increasethe surface area of the tissue over that in its naturally-occurringcondition. The conditioned tissue is then exposed to the aqueousextraction medium, with or without agitation, for a period of timesufficient to result in extraction of polypeptides and proteoglycans insubstantial yield, or until equilibrium conditions are attained.Extraction is desirably effected at temperatures below room temperature,and preferably at approximately 5° C.

Following extraction, the aqueous extract is treated to concentrate thegrowth inhibiting substance contained therein. It is believed that thegrowth inhibiting substance of the present invention is presentprimarily in the fraction of the extract having a molecular weight of50,000 or below. Accordingly, the extract may be treated so as tofractionate compounds having a molecular weight of 50,000 or less fromthose of higher molecular weight, by molecular sieve or ultrafiltrationtechniques, etc. or the like. The growth inhibiting substance of thepresent invention may be further concentrated by removal of water, as bylyophilization of the extract.

The growth inhibiting substance thus obtained has a marked inhibitoryeffect on the rate of proliferation of endothelial cells. It alsoinhibits proliferation of some fibroblasts, although inhibition ofgrowth of endothelial cells. In general, the growth inhibiting substanceof the present invention is more effective against the proliferation ofimmature cells than of mature cells.

The extraction medium employed in the practice of the present inventionmay be any aqueous extraction medium, except that the extraction mediumshould not irreversibly denature the proteinaceous matter extracted fromthe connective tissue. An aqueous extraction medium containing arelatively high salt content, i.e., 1.0-3.0 M, is desirably used. Apreferred extraction medium is a 1.0-3.0 M aqueous solution of guanidinehydrochloride.

If a high salt aqueous extraction medium is used, the salt should beremoved from the extract, and this may be accomplished by dialysis, inaccordance with known procedures.

The concentration of the growth inhibiting substance of the presentinvention in the connective tissue and in the aqueous extract is low.For example, it is necessary to extract several hundred grams ofconnective tissue in order to obtain a few milligrams of the lyophilizedextract having a molecular weight of 50,000 or below.

EXAMPLE I

As a specific example of the preparation of the growth inhibitingsubstance of the present invention, extracts were prepared from bovineconnective tissues. Bovine connective tissues which were extracted werenasal septum cartilage, scapular epiphyseal growth plate, and dermis.The tissues were minced and placed in five volumes of an extractionmedium comprising a 1.0 M aqueous solution of guanadine hydrochloride,the solution having a pH of 6.0. The mixture of tissue and extractionmedium was stirred for 24 hours at 5° C. The resultant aqueous extractwas separated from the tissue and dialyzed exhaustively against water.The dialyzed extract was then lyophilized.

Dialyzed and lyophilized extract from scapular epiphyseal growth platewas redissolved in 4.0 M guanidine hydrochloride solution, and dialyzedthrough an Amicon filter membrane with a pore size which allowedcompounds of molecular weight 50,000 or below to pass through themembrane. Following dialysis, the solutions on both sides of themembrane were dialyzed exhaustively against water and lyophilized.

There were thereby obtained lyophilized extracts of bovine cartilage andof bovine dermis, as well as lyophilized extracts from bovine cartilagewhich had been fractionated into an extract having a molecular weight of50,000 or below and an extract having a molecular weight higher than50,000. These lyophilized extracts were then used to demonstrate theiractivity as inhibitors of cell proliferation.

EXAMPLE II

Cultures of various tissue cell types were prepared for test purposes.Bovine aortic endothelial cells were cultured from aortas collected froma local slaughter house. Fresh aortas were immediately transported tothe laboratory. There, the two ends of each aorta were clamped and thebranches ligated. The lumen was rinsed with about 50 ml of 0.9% NaCl.The aorta was then slightly distended with 25-50 ml of complete tissueculture medium. After about 5 minutes, the fluid was removed with asyringe and needle and 5 ml aliquots dispensed into Falcon T-25 plasticflasks. The flasks were incubated at 37° C. in a humidified 5% Co₂ -airatmosphere. The cultures generally required 8-12 days to reachconfluency.

Steer fibroblasts were isolated from the subcutaneous tissue of earsfrom freshly slaughtered animals. Tissues were removed aseptically andincubated in a solution of 2 mg collagenase/ml HEPES. Two hours ofdigestion at 37° C. was sufficient to yield 1×10⁶ cells for plantinginto Falcon flasks.

Bovine embryo fibroblasts were purchased from GIBCO Corp., Grand Island,N.Y. Second passage human foreskin fibroblasts were also obtained.

The tissue culture medium used for all cell types was RPMI 1640,obtained from GIBCO Corp., Grand Island, N.Y., supplemented with 20%fetal calf serum which had been heated for 50 minutes at 56° C. toinactivate bovine infectious agents present in the serum. Increasedbuffering capacity was provided by adding 20 mM HEPES. Antibiotics wereadded to yield a final concentration of gentamycin, 50 μg/ml andamphotericin B, 5 μg/ml in the complete medium. The medium was made upfrom the commercially supplied powder as a 2X concentrate and thenpassed through 0.22μ pore size millipore filters for sterilization.

When tissue extracts or other materials were to be added, thelyophilized extracts were hydrated in distilled water, sterilized byfiltration, and then added to the concentrated culture medium. Thefiltration clarified the cloudiness seen in the aqueous solution of theextract.

For biological assays of the tissue extracts, established cultures ofsecondary cells were subcultured in 35×10 mm Falcon tissue culturedishes at an initial density of 2×10⁴ cells/ml in tissue culture mediumand an additional 1 ml of medium added. The next day, the medium wasremoved and replaced with 2 ml of tissue culture medium containing thematerials to be tested or with an equal volume of control medium.Control cell counts were done at this time.

The cultures were refed on the third day after test materials wereadded. A 2.25% solution of disodium ethylene diamine tetra-acetic acidwas then prepared in Ca-Mg free phosphate buffered saline, pH 7.4. Thecells were then removed from the dish by digesting with a 0.25% trypsinsolution. Cell counts of the trypsin dispersed cells were done with ahemocytometer after staining with a 1% filtered solution of tripan blue.Two culture dishes were counted each day for each experimentaltreatment. Each set of experiments was repeated at least four times. Atthe termination of the experiments, some cultures were fixed in 70%methanol and stained with hematoxylin. For dose-response curve studiesof cartilage-derived materials, counts were done only at the beginningof the experiment and 3 days after the test material was added. In suchexperiments, three dishes were counted for each dose and the experimentwas repeated three times.

Since identification of endothelial cells depends in large part on theWeibel-Palade bodies, electron microscopy was done on these culturedcells and these organelles were found as expected. For this, the culturedishes were washed with 0.9% NaCl. The cells were then scraped off theculture dish with a rubber spatula, fixed in 2.5% cacodylate buffered.[.gluteraldehyde.]. .Iadd.glutaraldehyde.Iaddend., washed overnight inbuffer, stained en bloc in uranyl acetate, dehydrated and embedded inEpon 812. Sections were mounted on copper grids, stained with leadcitrate and studied in an electron microscope.

The effects of the lyophilized extract of bovine cartilages on thegrowth of endothelial cells were determined. 40,000 endothelial cells in2 ml of culture medium were initially dispensed into 35 mm Petri dishes.The following day the extract to be tested was injected into the dish,three dishes for each extract. Three control dishes were also provided,which were refed only with culture medium the day after culturing wasbegun.

Three days after injection of the respective lyophilized extracts, cellcounts were done on each dish. In the case of the control dishes, theoriginal population of 40,000 cells had increased to 700,000. Table Ishows the cell counts of the dishes containing lyophilized cartilageextract at three different concentrations of lyophilized extract.

                  TABLE I                                                         ______________________________________                                        EFFECT OF CARTILAGE EXTRACTION                                                ENDOTHELIAL CELL GROWTH                                                       Dose,                Cells/dish                                               ______________________________________                                        micrograms lyophilized                                                        extract per ml. of culture                                                    medium                                                                        500                   52,000                                                  100                  285,000                                                   20                  510,000                                                   0                   700,000                                                  ______________________________________                                    

It will be seen from Table I that at a dosage of 500 micrograms oflyophilized extract per milliliter of culture medium, proliferation ofendothelial cells was substantially inhibited, the cell count increasingonly to 52,000 from the original count of 40,000. At lower dosages,inhibition was less complete, but in each case the cell count wassubstantially less than the 700,000 cell count of the control after 3days.

Similar testing was done utilizing the fraction of lyophilized cartilageextract having a molecular weight greater than 50,000. There was noobserved inhibition of proliferation of endothelial cells using thisfraction, the cell count at a dosage level of 500 micrograms/milliliterbeing 710,000 after three days, not substantially different from thecell count of 700,000 in the control.

However, the fraction of lyophilized cartilage extract having amolecular weight of 50,000 and below was substantially more potent as agrowth inhibitor than the lyophilized extract which was the subject ofTable I. Table II shows the results of this test, which was carried outin the same manner as testing of the unfractionated extract.

                  TABLE II                                                        ______________________________________                                        EFFECT OF CARTILAGE EXTRACT HAVING A                                          MOLECULAR WEIGHT OF 50,000                                                    AND BELOW ON ENDOTHELIAL CELL GROWTH                                          Dose                 Cells/dish                                               ______________________________________                                        micrograms lyophilized                                                        extract per ml. of culture                                                    medium                                                                        500                   22,000                                                  100                   65,000                                                   20                  260,000                                                   5                   300,000                                                   0                   700,000                                                  ______________________________________                                    

It will be seen from Table II that the fraction of lyopholized extracthaving a molecular weight of 50,000 or below was effective at inhibitingproliferation of endothelial cells at dosages as low as 5 micrograms permilliliter.

The tests of which the results are reported in Tables I and II wererepeated three times, and gave virtually identical results.

The same lyophilized extracts of bovine cartilage used in the tests ofwhich the results are shown in Tables I and II were evaluated foractivity in inhibiting proliferation of mature steer fibroblasts in thesame culture medium. There was no significant growth inhibition, now wasthere significant growth inhibition of steer fibroblasts by lyophilizedextracts of dermis. However, lyophilized extract from bovine cartilagewas found to inhibit proliferation of fetal bovine fibroblasts. Theinhibitory effect of lyophilized extract from bovine cartilage on fetalbovine fibroblasts was less marked than the inhibitory effect on bovineendothelial cells. There was also growth inhibition by extracts frombovine cartilage on infant human foreskin fibroblasts, but again thedegree of inhibition was less marked than in the case of endothelialcells.

Lyophilized extracts of dermis did not inhibit growth of fetal bovinefibroblasts, although they did inhibit growth of endothelial cells aseffectively as cartilage extracts. Lyophilized extracts of bovine aortaand of canine cartilage also inhibited endothelial cell growth.

The inhibitory effect of extracts from connective tissue on cellproliferation is believed to provide an explanation as to why some suchtissues are relatively resistant to invasion by either neoplasms orinflammatory processes. It has been observed for many years that poorlyvascularized or avascular tissues such as cartilage are relativelyresistant to invasion. Clinicians and pathologist have long known, forexample, that most forms of cancers of the respiratory passages oftenencase, but seldom invade, bronchial or laryngeal cartilages. Morerecently, investigation of the resistance of certain tissues to invasionby explantation on to the chick chorioallantoic membrane showed thattissues which normally have a blood supply are rapidly invaded byvascularized mesenchyme originating from the chick embryo. On the otherhand, post-natal hyaline cartilage, which is virtually devoid of bloodvessels, was substantially impenetrable under the same conditions.American Journal of Pathology, Volume 73, No. 3 Pages 765-772, December1973. Although applicants do not intend to be bound by theory, nor torestrict the scope of their invention by theory, it is believed that thepresence of the cell proliferation inhibiting substance of the presentinvention at relatively high concentrations in poorly vascularized oravascular tissues such as cartilage and blood vessels is responsible forthe resistance of such tissues to invasion.

EXAMPLE III

As a further specific example of the preparation of the growthinhibiting substance of the present invention, an extract was preparedfrom bovine cartilage utilizing an alternate extraction method. Bovinenasal septum cartilage was minced and placed in five volumes of anextraction medium comprising a 1.0 M aqueous solution of guanidinehydrochloride, the solution having a pH of 6.0 The mixture of tissue andextraction medium was stirred for 48 hours at 5° C.

The resultant aqueous extract was separated from the tissue, andguanidine hydrochloride was added to the aqueous extract in an amountsufficient to raise the concentration from 1.0 M to 3.0 M. The resultant3.0 M aqueous extract was subjected to pressure dialysis, using adialysis membrane which permitted passage through it of materials havinga molecular weight below about 100,000. Dialysis was continued untilequilibrium was attained. About 10% of the substance extracted from thetissue passed through the dialysis membrane.

The resultant dialysate comprising extracted substance having amolecular weight below about 100,000, was again subjected to pressuredialysis, using a membrane which permitted passage through it ofmaterials having a molecular weight of below about 50,000. The resultantdialysate was collected and dialysed exhaustively against water througha membrane to deplete it of guanidine hydrochloride and other substanceshaving a molecular weight below about 3,500. The dialyzed extract waslyophilized to provide a substance which also had activity as aninhibitor of cell proliferation.

Thus, there has been provided a composition of matter having activity asan inhibitor of cell proliferation, and there has also been providedmethods of preparing such a composition and methods of inhibiting cellproliferation utilizing such a composition.

Various of the features of the present invention are set forth in thefollowing claims.

What is claimed is:
 1. The method of preparing a composition of matterhaving activity as an inhibitor of cell proliferation .Iadd.and tissueinvasion .Iaddend.comprising the steps of providing connective tissuehaving a high content of collagen or proteoglycans in condition forextraction, extracting said inhibitor from said tissue with an aqueousextraction medium which includes a solute which does not irreversiblydenature the proteinaceous matter to be extracted, separating theresultant aqueous extract from the tissue, recovering from the aqueousextract substances having a molecular weight below about 50,000,treating the fraction of aqueous extract having a molecular weight belowabout 50,000 to remove salts therefrom, and dehydrating the resultantmaterial.
 2. The method of claim 1 wherein the connective tissuecomprises tissue normally devoid of an intimate capillary blood supply.3. The method of claim 2 wherein the tissue comprises cartilage.
 4. Themethod of claim 2 wherein the tissue comprises blood vessels.
 5. Acomposition of matter having activity as an inhibitor of cellproliferation .Iadd.and tissue invasion .Iaddend.comprising the productof the method of claim
 1. 6. A composition of matter having activity asan inhibitor of cell proliferation comprising the product of the methodof claim
 2. 7. A method of inhibiting proliferation of cells comprisingtreating the cells with the product of claim
 1. 8. A method ofinhibiting proliferation of cells comprising treating the cells with theproduct of claim
 2. 9. A method of inhibiting proliferation of cellscomprising treating the cells with the product of claim
 3. 10. A methodof inhibiting proliferation of cells comprising treating the cells withthe product of claim 4.